Treatment for adult-onset autosomal dominant leukodystrophy (ADLD)

Abstract

The present invention relates to a method for treating diseases or conditions associated with abnormal levels of lamin B1 protein. Disclosed herein is a method for treating conditions of lamin B1 accumulation, such as adult-onset autosomal dominant leukodystrophy (ADLD), using a farnesyltransferase inhibitor (FTI), such as FTI-277, lonafarnib, or tipifarnib.

Description

[Technical Field]

[0001] The present invention relates generally to methods for treating diseases or conditions associated with abnormal levels of lamin B1 protein. In particular, disclosed herein are methods for treating lamin B1 accumulation conditions, such as adult-onset autosomal dominant leukodystrophy (ADLD), using farnesyltransferase inhibitors (FTIs). [Background technology]

[0002] Disorders and mutations in the nuclear lamina and its components cause a wide variety of human diseases, collectively known as laminopathies, including cardiomyopathies, muscular dystrophies, lipodystrophies, and premature aging syndromes. While the majority of these diseases are caused by mutations in the lamin A gene (LMNA), overexpression of the lamin B1 gene (LMNB1) is also associated with certain pathologies. Lamins A, B1, and B2 undergo post-translational modification by the addition of a farnesyl moiety to their C-terminal cysteine ​​residues. In the case of lamin A, the 15 C-terminal amino acids, including the farnesyl tail, are subsequently cleaved to form the mature lamin A protein. Meanwhile, both lamins B1 and B2 retain the farnesyl group.

[0003] Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by central nervous system demyelination and age-dependent motor impairment. ADLD is a rare genetic disorder caused by duplication of the LMNB1 gene locus, resulting in elevated lamin B1 protein levels. Small variations in lamin B1 expression can have significant molecular and functional effects on cells of the nervous system, particularly the CNS. Patients typically begin to show symptoms, including motor and sensory impairments, in their mid-30s. Currently, there are no effective treatments available for ADLD or other laminopathies associated with abnormal lamin B1 levels. The contribution of lamin B1 farnesylation to the pathogenesis of these diseases is also unclear. Summary of the Invention [Problem to be solved by the invention]

[0004] It is therefore desirable to overcome or ameliorate at least one of the above-mentioned problems. [Means for solving the problem]

[0005] overview Disclosed herein is a method of inhibiting farnesylation of lamin B1 protein in a cell from a subject, comprising administering to the cell an effective amount of a farnesyltransferase inhibitor (FTI).

[0006] Disclosed herein is a method of inhibiting accumulation of lamin B1 protein in cells from a subject, comprising administering to the cells an effective amount of a farnesyltransferase inhibitor (FTI).

[0007] Disclosed herein are methods of treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein, comprising administering to the subject an effective amount of a farnesyltransferase inhibitor (FTI).

[0008] Disclosed herein are farnesyltransferase inhibitors for use in treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein.

[0009] Disclosed herein is the use of a farnesyltransferase inhibitor in the manufacture of a medicament for treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein.

[0010] Disclosed herein are methods of treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject, comprising administering to the subject an effective amount of a farnesyltransferase inhibitor (FTI).

[0011] Disclosed herein are farnesyltransferase inhibitors for use in treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject. Disclosed herein is the use of a farnesyltransferase inhibitor in the manufacture of a medicament for treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject.

[0012] Embodiments of the invention will now be described, by way of non-limiting example, with reference to the drawings in which: [Brief explanation of the drawings]

[0013] [Figure 1] Genetic inhibition of lamin B1 farnesylation prevents lamin B1 accumulation in human fibroblasts. Wild-type v5-LB1 CAIM or a farnesylatable mutant of lamin B1 (v5-LB1 SAIM) was expressed in normal (WT) or lamin A / C (shLA / C) human fibroblasts upon DOX addition. Western blot analysis showed reduced accumulation of v5LB1 SAIM in both WT and shLA / C fibroblasts. Actin was used as a loading control. [Figure 2] Treatment with three different FTIs prevents lamin B1 accumulation. (A) Schematic of the experimental design for panels (B) and (C) using the inducible LB1 NDF cell line. (B) Representative Western blots of NDF expressing LB1 over 3 days in the presence of DMSO control, FTI-277, lonafarnib, and tipifarnib. Time after v5, GAPDH, LA / C, and DOX induction is indicated. (C) Quantification of three independent sets shown in (B). Protein levels were normalized to GAPDH relative to 8-hour v5 levels in the DMSO control (n = 3, two-way ANOVA with Bonferroni post-hoc test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). [Figure 3]FIG. 1 shows doxycycline-induced expression of lamin B1 over 72 hours with DMSO or lovastatin. [Figure 4] FTI-277 specifically reduces LB1 accumulation. (A) Schematic of both LB1 constructs (LB1 and LB1 SAIM) expressed under a DOX-inducible promoter. The SAIM mutation prevents LB1 farnesylation by substituting a cysteine ​​in the CaaX box. (B) Schematic of the experimental design for (C) and (D) using the cell lines indicated in (A). (C) Representative Western blots of NDFs expressing LB1 or LB1 SAIM for 16 days in the presence of DMSO control or FTI-277. v5, GAPDH, LA / C, and days after DOX induction are indicated. (D) Quantification of three independent sets shown in (C). Protein levels were normalized to GAPDH relative to day 4 v5 levels in the DMSO control (n = 3, two-way ANOVA with Bonferroni post-hoc test; *≤0.05, **≤0.01, ***≤0.001). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present specification teaches a method for inhibiting farnesylation of lamin B1 protein. The present specification discloses a method for inhibiting farnesylation of lamin B1 protein in cells derived from a subject, comprising administering an effective amount of a farnesyltransferase inhibitor (FTI) to the cells. In one embodiment, inhibiting farnesylation of lamin B1 inhibits the accumulation of lamin B1 protein in the cells. In another embodiment, inhibiting farnesylation of lamin B1 reduces the level of lamin B1 in the cells.

[0015] Without being bound by theory, the present inventors have discovered that genetically blocking farnesylation of lamin B1 slows the intracellular accumulation of the protein and can be used to treat lamin B1-related diseases such as adult-onset autosomal dominant leukodystrophy (ADLD). The present inventors have further discovered that farnesyltransferase inhibitors (FTIs) can be used to block lamin B1 accumulation. Thus, FTIs can be used to treat diseases caused by abnormally elevated levels of lamin B1 or other constitutively farnesylated proteins.

[0016] Also disclosed herein is a method of inhibiting accumulation of lamin B1 protein in a cell from a subject, comprising administering to the cell an effective amount of a farnesyltransferase inhibitor (FTI).

[0017] Also disclosed herein is a method of reducing the level of lamin B1 protein in a cell from a subject, comprising administering to the cell an effective amount of a farnesyltransferase inhibitor (FTI).

[0018] In one embodiment, the subject suffers from a disease or condition associated with an abnormal level of lamin B1 protein. Such diseases or conditions are generally characterized by an abnormal intracellular level of lamin B1 protein. For the avoidance of doubt, an abnormal level of lamin B1 does not necessarily have to be the causative factor of the disease or condition, and may be, for example, secondary to other genetic and / or cellular defects that also exist in the disease or condition. The term "associated with" includes not only the disease or condition itself, but also an increased risk of developing the disease or condition. Thus, for example, an abnormal increase in lamin B1 level may be associated with ADLD and the tendency to develop ADLD.

[0019] The term "abnormal level" refers to a chronic or progressive change in the level of a protein that results in a disease or pathological condition. An abnormal level of a protein can be determined by comparison with a normal level or reference level. This normal level or reference level can be found in a control, standard, population standard, etc. For example, if an abnormal protein level is associated with a disease, the normal level or reference level can be the protein level in cells of an individual who is not affected by the disease (or the average protein level in a population of cells), or a level that is a population standard for individuals who are not considered to be affected by the disease, etc.

[0020] The abnormal level can be an abnormally high or an abnormally low level of Lamin B1. The Lamin B1 level can be abnormal in one or more cell types, tissues or organs in the subject.

[0021] The terms "elevated levels" and "accumulation" are used interchangeably herein to refer to abnormally high protein levels. Lamin B1 levels may be abnormally high if they exceed a reference level by, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, or more.

[0022] The terms "reduced levels" and "decreased" are used interchangeably herein to refer to abnormally low protein levels. Lamin B1 levels may be abnormally low if they are, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% below a reference level.

[0023] Abnormal lamin B1 levels may also be due to abnormal nucleic acids, such as abnormal LMNB1 nucleic acids encoding lamin B1. Such abnormalities include, but are not limited to: (1) mutations in nucleic acids, such as point mutations (e.g., single nucleotide polymorphisms) or deletions, duplications, additions, or inversions of nucleotide sequences; (2) mutations in regulatory sequences associated with the nucleic acid, such that replication or expression of the nucleic acid is altered (e.g., functional inactivation of a promoter); (3) changes in the amount or copy number of the nucleic acid in a cell compared to a control or standard (e.g., nucleic acid duplication or genomic amplification, or overexpression of mRNA); and (4) changes in sequences controlling the splicing machinery, such that normal splice signals are inactivated or abnormal splice signals are generated. It is understood that these types of abnormalities can coexist within the same nucleic acid or the same cell; for example, a genomically amplified nucleic acid sequence may contain one or more point mutations. Furthermore, it is understood that nucleic acid abnormalities can be associated with and actually cause abnormal expression of the corresponding protein.

[0024] Abnormalities in lamin B1 levels may also be due to abnormalities in protein expression, including, but not limited to, (1) mutations in the protein that result in one or more different amino acid residues; (2) deletions, additions, or duplications of a series of amino acids in the protein's sequence; (3) increased or decreased expression of the protein compared to a control or standard; (4) changes in the subcellular localization or targeting of the protein; (5) changes in the temporal expression of the protein (such that the protein is expressed when it would not normally be expressed, or is not expressed when it would normally be expressed); and (6) changes in the localized (e.g., organ- or tissue-specific) expression of the protein (such that the protein is not expressed where it would normally be expressed, or is expressed where it would not normally be expressed), respectively, compared to a control or standard.

[0025] Another type of protein abnormality contemplated herein includes alterations in post-translational processing of proteins. Proteins with abnormal post-translational processing include, for example, proteins that are processed in a manner or to a different extent than wild-type (normal) versions of the protein. For example, wild-type lamin B1 protein is constitutively or constitutively farnesylated, and lamin B1 protein lacking a farnesyl group (e.g., lamin B1 lacking a farnesylation site) is considered a farnesylation abnormal form of lamin B1. Other potential post-translational processing abnormalities include alterations in other attachment groups, such as methylation, phosphorylation, glycosylation, etc.

[0026] In one embodiment, the subject suffers from a disease or condition associated with the accumulation of lamin B1 protein.Non-limiting examples of diseases or conditions associated with the accumulation of lamin B1 include adult-onset autosomal dominant leukodystrophy (ADLD), Werner syndrome, ataxia-telangiectasia, Huntington's disease, and cancers, including but not limited to pancreatic cancer, liver cancer, and ovarian cancer.

[0027] In one embodiment, the subject suffers from adult-onset autosomal dominant leukodystrophy (ADLD).ADLD is characterized by progressive demyelination in the subject's brain and spinal cord, resulting in autonomic nervous system dysfunction and eventual motor disability in adulthood.ADLD is a genetic disease caused by duplication of lamin B1 gene (LMNB1) or deletion near the LMNB1 locus, resulting in excessive lamin B1 in cells.Therefore, FTI is particularly suitable for treating ADLD by inhibiting the accumulation of lamin B1.

[0028] As used herein, the term "subject" refers to a mammal. A subject can be a human or a non-human mammal such as a dog, cat, cow, horse, mouse, rat, rabbit, or transgenic species thereof. A subject can be a patient. In one embodiment, the subject is a human.

[0029] The cell can be any cell that expresses Lamin B1 protein, including, but not limited to, cells of the central and peripheral nervous system, musculoskeletal system, digestive system, cardiovascular system, lymphatic system, respiratory system, integumentary system, gastrointestinal system, male and female reproductive system, endocrine system, and urinary system.

[0030] In one embodiment, the cell is a cell of the central nervous system or peripheral nervous system. The cell may be, for example, a neuron, oligodendrocyte, astrocyte, ependymal cell, microglial cell, Schwann cell, satellite cell, or immune or vascular cell of the central nervous system or peripheral nervous system. The cell may be involved in forming the myelin sheath around neurons, such as an oligodendrocyte or astrocyte of the central nervous system or a Schwann cell of the peripheral nervous system.

[0031] In one embodiment, the cells have abnormal levels of Lamin B1 protein, which may be abnormally high or abnormally low levels of Lamin B1. In one embodiment, the cells have accumulated Lamin B1 protein.

[0032] As used herein, the term "farnesyltransferase inhibitor (FTI)" refers to a substance that reduces the expression or activity of farnesyltransferase. The reduction in farnesyltransferase expression or activity may be cell-, tissue-, or organ-specific, and may be transient or permanent.

[0033] Farnesyltransferase is an enzyme that transfers a farnesyl group from farnesyl diphosphate to a cysteine ​​residue in a CAAX box motif (C: cysteine, A: aliphatic amino acid, X: any amino acid) in proteins. This enzyme is a heterodimer consisting of two subunits: an α-subunit encoded by the FNTA gene and a β-subunit encoded by the FNTB gene. The α-subunit is also found in geranylgeranyltransferase and is required for the structural stability of the farnesyltransferase enzyme. The β-subunit is responsible for substrate binding and catalysis. As a non-limiting example, FTIs can target one or both enzyme subunits, interactions between subunits, or interactions between the enzyme and non-substrate proteins, cells, or intracellular components.

[0034] Substances that reduce farnesyltransferase activity include, but are not limited to, small molecules, peptides (e.g., allosteric antagonists or substrate mimetics), proteins (e.g., antibodies), nucleic acids (e.g., aptamers), and conjugates of two or more of these. Inhibitors may also be nucleic acids that encode inhibitory peptides or polypeptides. Inhibitors can inhibit enzyme activity directly (e.g., by preventing interaction with the substrate or by altering the structure of the enzyme) or indirectly (e.g., by affecting the intracellular localization of the enzyme or the interaction of the enzyme with cofactors or subunits).

[0035] Substances that inhibit the expression of farnesyltransferase may act on the LMNB1 gene (e.g., by selectively altering the nucleic acid sequence of the gene, thereby preventing gene expression and / or producing a non-functional enzyme), a promoter operably linked to the LMNB1 gene (e.g., by preventing or reducing gene transcription), or the RNA product of the LMNB1 gene (e.g., by degrading or silencing lamin B1 mRNA). The inhibitor may inhibit transcription and / or translation of the gene from the RNA product of the gene, induce exon skipping, otherwise interfere with RNA splicing, or mediate sequence-specific cleavage, degradation, or editing of LMNB1 or the RNA product of the gene. The inhibitor may be a nucleic acid molecule, such as a single-stranded or double-stranded DNA or RNA molecule. The inhibitor may mediate inhibition by itself or together with a polypeptide or another nucleic acid molecule. Furthermore, the inhibitor undergoes biochemical or enzymatic processing after administration (e.g., in the cells or tissues of the subject) to become functionally inhibited.

[0036] In one embodiment, the FTI inhibits farnesyltransferase expression. In one embodiment, the FTI is a nucleic acid inhibitor. Non-limiting examples of nucleic acid inhibitors include antisense compounds (e.g., antisense oligonucleotides (ASOs), gapmers, etc.); nucleic acid molecules that mediate RNA interference (e.g., small segmented siRNAs, small interfering ribonucleic neutrals, Dicer substrate siRNAs, etc.), including, but not limited to, short hairpin RNAs (shRNAs), small interfering RNAs (siRNAs), and their variants and precursors, as well as microRNAs (miRNAs) and precursors; and guide RNAs (gRNAs) that mediate sequence-specific base editing, gene or RNA editing, or DNA or RNA cleavage in association with Cas proteins. The inhibitor may be a vector (e.g., a plasmid vector, a viral vector, etc.) encoding the nucleic acid inhibitor.

[0037] The terms "polynucleotide," "genetic material," "genetic forms," ​​"nucleic acid," and "nucleotide sequence" include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, as well as both sense and antisense strands, which may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art.

[0038] The term "nucleotide" refers to ribonucleotides, deoxyribonucleotides, or modified forms thereof, and analogs thereof. Nucleotides include species containing purines, such as adenine, hypoxanthine, guanine, and their derivatives and analogs, and pyrimidines, such as cytosine, uracil, thymine, and their derivatives and analogs.

[0039] Nucleotide analogs include nucleotides with modifications to the chemical structure of the base, sugar, and / or phosphate, including, but not limited to, 5-position pyrimidine modifications, 8-position purine modifications, modifications with the exocyclic amine of cytosine, and 5-bromo-uracil substitution; and 2'-position sugar modifications, including, but not limited to, sugar-modified ribonucleotides in which the 2'-OH is replaced with a group such as H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Nucleotide analogs are also meant to include nucleotides with bases such as inosine, querceine, and xanthine, sugars such as 2'-methylribose, and non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates, and peptides.

[0040] Modified base refers to the nucleotide base, such as adenine, guanine, cytosine, thymine, uracil, xanthine, inosine and quercein, which is modified by replacing or adding one or more atoms or groups.Some examples of the types of modifications that can be made for the modified nucleotide of base moiety include, but are not limited to, alkylation, halogenation, thiolation, amination, amidation or acetylation, individually or in combination. More specific examples include 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine, and other nucleotides modified at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, 7-deazaadenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine, etc. deazanucleotides, other thio bases such as 2-thiouridine, 4-thiouridine, 2-thiocytidine, dihydrouridine, pseudouridine, queosine, archaeosine, naphthyl and substituted naphthyl groups, N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, any O- and N-alkylated purines and pyrimidines such as pyridin-4-one, pyridin-2-one, phenyl groups, and modified phenyl groups such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides.Modified nucleotides also include nucleotides modified with respect to the sugar moiety, as well as nucleotides having non-ribosyl sugars or analogs thereof, for example, the sugar moiety can be or be based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles.

[0041] In one embodiment, the FTI inhibits farnesyltransferase activity. The FTI may be a small molecule. Non-limiting examples of FTIs that inhibit farnesyltransferase activity include chaetomellic acid A, clavaric acid, FPT inhibitor I, FPT inhibitor II, FPT inhibitor III, FTase inhibitor I, FTase inhibitor II, FTI-276 trifluoroacetate, FTI-277 trifluoroacetate, GGTI-297, L-744,832 dihydrochloride, manumycin A, gingerol, gliotoxin, α-hydroxyfarnesylphosphonic acid, tipifarnib, lonafarnib (SCH-66336), CP-609,754, BMS-214662, L778123, L744823, L739749, R208176, AZD3409, or FTI-277. In one embodiment, the FTI is FTI-277, lonafarnib, or tipifarnib.

[0042] In some embodiments, an FTI reduces lamin B1 farnesylation by at least about 10%. An FTI can reduce lamin B1 farnesylation by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. An FTI can reduce lamin B1 farnesylation by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0043] In some embodiments, an FTI reduces lamin B1 accumulation by at least about 10%. An FTI can reduce lamin B1 accumulation by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. An FTI can reduce lamin B1 accumulation by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0044] In some embodiments, an FTI reduces lamin B1 levels by at least about 10%. An FTI can reduce lamin B1 levels by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%. An FTI can reduce lamin B1 levels by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0045] The level of lamin B1 protein and the level of lamin B1 farnesylation can be determined using methods known in the art, for example, immunological methods such as immunocytochemistry or Western blotting using labeled antibodies that bind to the farnesyl group or lamin B1. Mass spectrometry can also be used to quantitate the levels of lamin B1 and farnesylation in cells.

[0046] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in terms of a partial or complete cure of a disease or condition and / or a partial or complete cure of side effects caused by a disease or condition. These terms also encompass any treatment of a condition or disease in a mammal, particularly a human, including: (a) inhibiting the disease or condition, i.e., arresting its onset; or (b) relieving the disease or condition, i.e., causing regression of the disease or condition.

[0047] "Effective amount" in the context of treating or preventing a disease or condition means that an amount of an active agent effective for treating or preventing the disease or condition is administered to a subject as a single dose or as part of a continuous dose or sustained release system.The effective amount varies depending on the subject's health and physical condition, the taxonomic group of the individual to be treated, the severity of the disease or condition, the formulation of the active agent or pharmaceutical composition, the evaluation of the medical condition, and other relevant factors.Those skilled in the art will be able to determine the effective amount of an active agent, taking into account, for example, the subject's age, weight, clinical condition, etc.

[0048] Disclosed herein is a method for identifying a subject who is likely to respond to treatment with a farnesyltransferase inhibitor (FTI), comprising detecting the level of lamin B1 in a sample from the subject, wherein an increase in the level of lamin B1 compared to a reference indicates that the subject is likely to respond to the farnesyltransferase inhibitor (FTI).

[0049] As used herein, "sample" includes any biological specimen that can be extracted, unprocessed, processed, diluted, or concentrated from a subject. Samples include collections of similar fluids, cells, or tissues isolated from a subject (e.g., surgically removed tissue, biopsy including fine needle aspiration), as well as fluids, cells, or tissues present within a subject. Any suitable method for obtaining a biological sample can be employed; exemplary methods include, for example, bloodletting, swabbing (e.g., buccal swabbing), fine needle aspiration, and forceps biopsy. Samples may also be pooled from multiple aliquots.

[0050] As used herein, a "reference," "control," "reference sample," or "control sample" refers to a sample, cell, tissue, standard, or level used for comparison purposes. In one embodiment, the reference is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of the same subject or individual. For example, a healthy and / or non-diseased cell or tissue adjacent to a diseased cell or tissue. In another embodiment, the reference is obtained from untreated tissue and / or cells of the body of the same subject or individual. In yet another embodiment, the reference is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of an individual other than the subject or individual. In another embodiment, the reference is obtained from untreated tissue and / or cells of the body of an individual other than the subject or individual. The reference may be the population average level of a biomarker (e.g., lamin B1) in healthy cells or tissues.

[0051] As used herein, the terms "increase" or "elevation" with respect to a biomarker, such as lamin B1, may refer to a statistically significant and measurable increase in the biomarker compared to a reference. The increase can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, or more.

[0052] In some embodiments, the subject is afflicted with adult-onset autosomal dominant leukodystrophy (ADLD). Disclosed herein is a method for treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein, comprising the steps of: a) detecting the level of lamin B1 in a sample from the subject, wherein an increase in the level of lamin B1 compared to a reference indicates that the subject is likely to respond to a farnesyltransferase inhibitor (FTI); and b) administering an effective amount of an FTI to a subject found to be likely to respond to an FTI.

[0053] In some embodiments, the disease or condition is adult-onset autosomal dominant leukodystrophy (ADLD). Disclosed herein are methods of treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein, comprising administering to the subject an effective amount of a farnesyltransferase inhibitor (FTI).

[0054] In some embodiments, the disease or condition is characterized by abnormal farnesylation of lamin B1 protein. Abnormal farnesylation refers to an alteration in lamin B1 farnesylation that results in abnormally high levels of lamin B1. Such an alteration can be, for example, an increase in farnesylation efficiency.

[0055] In some embodiments, the disease or condition is characterized by accumulation of lamin B1 protein. In one embodiment, the disease or condition is adult-onset autosomal dominant leukodystrophy (ADLD).

[0056] Disclosed herein are methods of treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject, comprising administering to the subject an effective amount of a farnesyltransferase inhibitor (FTI).

[0057] Also disclosed herein is a method for treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject, the method comprising: a) detecting a level of lamin B1 in a sample from the subject, wherein an increase in the level of lamin B1 compared to a reference indicates that the subject is likely to respond to a farnesyltransferase inhibitor (FTI); and b) administering an effective amount of an FTI to a subject found to be likely to respond to an FTI.

[0058] In one embodiment, an FTI is provided for use in treating a disease or condition in a subject associated with abnormal levels of Lamin B1 protein. In one embodiment, an FTI is provided for use in treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject.

[0059] In one embodiment, there is provided the use of an FTI in the manufacture of a medicament for treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein. In one embodiment, there is provided the use of an FTI in the manufacture of a medicament for treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject.

[0060] The FTIs defined herein can be administered in a single dose or in a series of doses. While it is possible to administer the active ingredient alone, it is preferable to present it as a composition, preferably as a pharmaceutical composition. The formulation of such compositions is well known to those skilled in the art. The composition may contain any suitable carrier, diluent, or excipient. These include all conventional solvents, dispersion media, fillers, solid carriers, coating agents, antifungal and antibacterial agents, transdermal penetration agents, surfactants, isotonic and absorption agents, etc. It will be understood that the compositions of the present invention may also contain other supplementary physiologically active agents.

[0061] In one embodiment, a pharmaceutical composition is provided comprising an FTI and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" means a pharmaceutical vehicle composed of a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject together with a selected active agent without causing any or substantial adverse reaction. Carriers may include excipients and other additives such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, and the like.

[0062] Carrier must be pharmaceutically " acceptable " in the sense that it is compatible with other components of composition and is not harmful to patient.Composition can be conveniently presented in unit dosage form and can be prepared by any method known in the art of pharmacy.This method includes the step of associating active ingredient with carrier that constitutes one or more accessory ingredients.Generally, composition is prepared by uniformly and intimately associating active ingredient with liquid carrier or finely divided solid carrier or both, and then, if necessary, shaping product.

[0063] Representative pharmaceutically acceptable carriers include any solvent, dispersion medium, coating agent, surfactant, antioxidant, preservative (e.g., antibacterial agent, antifungal agent), isotonicity agent, absorption retardant, salt, preservative, drug, drug stabilizer, gel, binder, excipient, disintegrant, lubricant, sweetener, flavoring agent, dye, and such materials and their combinations that will be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, pp. 1289-1329, which is incorporated herein by reference).Unless any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

[0064] Pharmaceutical compositions may be in various forms. For example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories, are included. The preferred form depends on the intended mode of administration and therapeutic use. Suitable pharmaceutical compositions can be administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, for example, orally, sublingually, bucally, sublingually, nasally, rectally, vaginally, or via pulmonary routes. In one embodiment, the mode of administration is oral. In one embodiment, the mode of administration is parenterally (e.g., intravenously, subcutaneously, intraperitoneally, intramuscularly).

[0065] As used herein, the phrases "parenteral administration" and "parenterally administered" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0066] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. In the subject invention, pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, preferably 0.05 M, phosphate buffer or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, inert gases, and the like.

[0067] More specifically, pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the compositions must be sterile and fluid to the extent that easy syringability exists. They should also be stable under the conditions of manufacture and storage, preferably preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin and / or by maintaining the required particle size. In specific embodiments, the FTIs of the present disclosure can be conjugated to a vehicle for cellular delivery. In these embodiments, the FTIs can be encapsulated in an appropriate vehicle to aid in delivery of the FTI to target cells, increase the stability of the FTI, or minimize potential toxicity of the FTI. As will be understood by those skilled in the art, various vehicles are suitable for delivering the FTIs of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems. Methods for incorporating the FTIs of the present disclosure into delivery vehicles are known in the art. Although various embodiments are presented below, it will be understood that other methods known in the art for incorporating the FTIs of the present disclosure into delivery vehicles are contemplated.

[0068] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. The FTIs of the present disclosure may be administered multiple times. The intervals between single doses may be daily, weekly, monthly, or yearly. The intervals may also be irregular, as indicated by measuring the patient's blood levels of the FTI. Alternatively, the FTI may be administered as a sustained-release formulation, requiring less frequent administration. The dosage and frequency of administration will vary depending on the half-life of the FTI in the patient.

[0069] For ease of administration and uniformity, it is particularly advantageous to formulate the composition in dosage unit form.Dosage unit form as used herein refers to a physically discrete unit that is suitable as a unit dose for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in association with necessary pharmaceutically acceptable carrier.The specification of dosage unit form of the present invention is determined and directly depends on (a) the inherent characteristics of active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the technology of compounding this active compound for the treatment of individual sensitivity.

[0070] In some embodiments, an effective amount of FTI is administered orally or parenterally. In some embodiments, FTI is administered orally in an amount of 1 to up to 1500 mg / kg per day, or more specifically, in an amount of 10 to 1200 mg / kg per day, as a single dose or divided into multiple doses. In some embodiments, FTI is administered orally in an amount of 100 mg / kg per day, 200 mg / kg per day, 300 mg / kg per day, 400 mg / kg per day, 500 mg / kg per day, 600 mg / kg per day, 700 mg / kg per day, 800 mg / kg per day, 900 mg / kg per day, 1000 mg / kg per day, 1100 mg / kg per day, or 1200 mg / kg per day.

[0071] It should be noted that dosage values ​​can vary depending on the type and severity of the condition to be alleviated.Furthermore, for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and it should be understood that dosage ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0072] In one embodiment, the pharmaceutical combination of FTI and another therapeutic agent is provided.The administration of the pharmaceutical combination of the present invention can bring about beneficial effects, such as additive or synergistic therapeutic effects, as well as other surprising beneficial effects, such as symptom alleviation, delaying or inhibiting progression.Such other effects include fewer side effects, improved quality of life, or reduced morbidity compared with the monotherapy of only one of the pharmaceutically active ingredients used in the combination of the present invention.

[0073] A further advantage of the present invention is that the active ingredients of the combination can be used in lower doses, which not only need to be administered less frequently but can also reduce the incidence or severity of side effects.

[0074] In particular, a therapeutically effective amount of each of the combination partners of the combination of the present invention may be administered simultaneously or sequentially in any order, and the components may be administered separately or as a fixed combination.

[0075] The effective dosage of each of the combination partners employed in the combination of the present invention can vary depending on the specific compound or pharmaceutical composition employed, the mode of administration, the condition being treated and the severity of the condition being treated.Therefore, the administration regimen of the combination of the present invention is selected according to various factors, including the route of administration and the patient's renal and hepatic function.Those skilled in the art can easily determine the effective amount of each single active ingredient required to alleviate, counter or prevent the progression of the condition.

[0076] As used herein, "and / or" refers to any and all possible combinations of one or more of the associated listed items and also encompasses the absence of combinations when interpreted as alternatives (or).

[0077] As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

[0078] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a stated integer or step or group of integers or steps, but not to the exclusion of other integers or steps or groups of integers or steps.

[0079] Any reference herein to any prior publication (or information derived therefrom) or known matter should not be construed as an acknowledgement, admission, or any form of suggestion that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

[0080] Those skilled in the art will understand that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications within its spirit and scope. The invention also includes all steps, features, compositions, and compounds referred to or shown in this specification, individually or collectively, and any combination of any two or more of said steps or features.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0082] Specific embodiments of the present invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the generality set forth hereinabove. [Example]

[0083] Materials and Methods cell culture The NDF cell line was provided by the Asian Skin Biobank (A*STAR) and cultured in NDF medium (Invitrogen, 10370088) supplemented with 15% fetal bovine serum (Invitrogen, SH30071.03), 2 mM glutamine (Invitrogen, 25030081-P), 0.2 mM non-essential amino acids (Invitrogen, 10370088), and 50 U / ml penicillin-streptomycin (Invitrogen, 15140122). Standard culture conditions (37°C, 5% CO2) were used. When subcultured, cells were always subcultured using 0.25% trypsin containing EDTA (Gibco, 25200056) and neutralized with dPBS (Cytiva, SH30028.02) containing 10% fetal bovine serum (Invitrogen, SV30160.03). Working concentrations of 1 μg / ml DOX (Clontech, 631311) and 5 μM FTI-277 (obtained from A*SRL) were used for the indicated experiments.

[0084] Treatment with statins and FTIs NDFs containing DOX-induced LB1 were treated with 5 μM lovastatin, 5 μM FTI-277, 2 μM lonafarnib, and 1 μM tipifarnib.

[0085] Western blotting Protein lysates were extracted using the cOmplete™ Lysis-M EDTA-Free Kit (Roche, 4719964001), cOmplete™ Protease Inhibitor Cocktail (Roche, 4693159001), 2% SDS (Promega, V6551), and 0.1 mM dithiothreitol (Sigma, 646563) according to the manufacturer's protocol. Protein quantification was performed using the Pierce™ Microplate BCA Protein Assay Kit - Reducing Agent Compatible (Thermo Scientific™, 23252). SDS-PAGE was performed in NuPAGE™ MOPS SDS (Invitrogen, NP0001) using 4-12% Bis-Tris gels (Invitrogen). Proteins were then transferred from the gel to a nitrocellulose membrane (Bio-Rad, 1620115) and subsequently blocked with Intercept™ (PBS) blocking buffer (Li-COR, LIR.927-70001) for 1 hour. The membrane was stained with primary antibodies overnight at 4°C, washed with PBS containing 0.1% Tween-20, and stained with Odyssey infrared-labeled secondary antibodies (LI-COR) at room temperature in the dark. Blots were visualized using a LI-COR Odyssey scanner. The resulting integrated intensities were normalized to GAPDH or actin loading control signals and analyzed using Microsoft Excel.

[0086] antibody The following primary antibodies were used: v5 (Abcam; ab9137, and Invitrogen, R960-CUS); lamin B1 (ProteinTech, 66095-1 Ig); LA / C (Millipore; MAB3211, and ProteinTech, 10298-1-AP); GAPDH (Sigma; G9545, and ProteinTech; 10494-1-AP). Secondary antibodies for Western blot were as follows: IRDYE 680 donkey anti-mouse IgG (Li-COR; 926-32222); IRDYE 800 donkey anti-mouse IgG (Li-COR; 926-32212); IRDYE 680 donkey anti-rabbit IgG (Li-COR; 926-68073); IRDYE 800 donkey anti-rabbit IgG (Li-COR; 926-32213); IRDYE 680 donkey anti-goat IgG (Li-COR; 926-32224); and IRDYE 800 donkey anti-goat IgG (Li-COR; 925-32214).

[0087] Example 1 To investigate whether the farnesyl group is involved in the pathogenesis of ADLD, we used a doxycycline-inducible system to ectopically express v5-tagged lamin B1 and a farnesylatable mutant of lamin B1 (in which the farnesylatable C-terminal cysteine ​​was changed to serine, v5-LB1-CAIM → v5-LB1-SAIM) in normal and lamin A / C-reduced (shLA / C) human primary neonatal dermal fibroblasts (NDF cells). Cells were treated with DOX in the presence of lovastatin and various FTIs for 3 days (Figures 1-3) or 16 days (Figure 4). We observed that a non-farnesylatable version of lamin B1 (v5-lamin B1 SAIM) did not accumulate as rapidly as the wild-type lamin B1 (v5-lamin B1 CAIM) protein (Fig. 1 ), suggesting that the farnesyl group increases lamin B1 stability and that farnesylation of lamin B1 is required for lamin B1 accumulation.

[0088] Example 2 To investigate whether pharmacological blocking of farnesylation with farnesyltransferase inhibitors (FTIs) could recapitulate these findings, we expressed v5-lamin B1 in the presence or absence of three FTIs (FTI-277, lonafarnib, and tipifarnib) and lovastatin (a drug that reduces both isoprenoid and cholesterol synthesis). All four drugs significantly delayed lamin B1 accumulation over 72 hours (Figures 2 and 3) and 16 days (Figure 4). These results indicate that farnesylation plays an important role in regulating LB1 levels and can be modulated by FTIs. Because ADLD patients have elevated LB1 levels due to duplication of the LMNB1 locus and no known treatment options, these findings strongly suggest that FTIs could be used to downregulate LB1 levels in these patients. Furthermore, FTIs may be useful for treating, preventing, or delaying the onset of diseases characterized by the accumulation of constitutively farnesylated proteins.

Claims

1. A method for inhibiting farnesylation of lamin B1 protein in a cell derived from a subject, comprising administering to the cell an effective amount of a farnesyltransferase inhibitor (FTI).

2. The method of claim 1, wherein the cells have an abnormal level of lamin B1 protein.

3. 3. The method of claim 2, wherein the subject is afflicted with adult-onset autosomal dominant leukodystrophy (ADLD).

4. 4. The method of claim 1, wherein inhibition of farnesylation of lamin B1 inhibits accumulation of lamin B1 protein.

5. 5. The method of any one of claims 1 to 4, wherein the cell is a cell of the central nervous system or peripheral nervous system.

6. The method of any one of claims 1 to 5, wherein the FTI is FTI-277, lonafarnib, or tipifarnib.

7. The method of any one of claims 1 to 6, wherein the subject is a human.

8. A method for inhibiting accumulation of lamin B1 protein in cells derived from a subject, comprising administering to the cells an effective amount of a farnesyltransferase inhibitor (FTI).

9. A method of treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein, comprising administering to the subject an effective amount of a farnesyltransferase inhibitor (FTI).

10. 10. The method of claim 9, wherein the disease or condition is characterized by accumulation of lamin B1 protein.

11. 11. The method of claim 9 or 10, wherein the disease or condition is adult-onset autosomal dominant leukodystrophy (ADLD).

12. The method of any one of claims 9 to 11, wherein the FTI is FTI-277, lonafarnib, or tipifarnib.

13. 13. The method of any one of claims 9 to 12, wherein the subject is a human.

14. A farnesyltransferase inhibitor for use in treating a disease or condition in a subject associated with abnormal levels of lamin B1 protein.

15. 10. Use of a farnesyltransferase inhibitor in the manufacture of a medicament for treating a disease or condition in a subject associated with an abnormal level of lamin B1 protein.

16. A method of treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject, comprising administering to the subject an effective amount of a farnesyltransferase inhibitor (FTI).

17. 17. The method of claim 16, wherein the ADLD is associated with abnormal levels of lamin B1 protein.

18. A farnesyltransferase inhibitor for use in treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject.

19. 1. Use of a farnesyltransferase inhibitor in the manufacture of a medicament for treating adult-onset autosomal dominant leukodystrophy (ADLD) in a subject.

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